Sterile protection against P. vivax malaria by repeated blood stage infection in the Aotus monkey model

Repeated blood stage infections with the human malaria parasite, Plasmodium vivax, induce sterile strain-specific and partial strain-transcendent protection in the Aotus monkey model.


General Statements [optional]
The two reviewers are very positive and emphasize the relevance of this study.Reviewer 1 notes that "the humoral immune responses but also parasite transcriptomics data is examined for the first time".Reviewer 2 notes that our study "tries to mimic the infection in nature by reinfecting the Aotus monkeys with different stains of the parasite and then assesses the immune response with main emphasis on antibody response to the infection.This model is important to facilitate vaccine development and understanding the immune response against particular vaccines." Both reviewers only suggest text changes.

Point-by-point description of the revisions
This section is mandatory.Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript.
Below please find the comments from reviewers 1 and 2 and our responses in italic, including text changes in the manuscript.
Reviewer #1 (Evidence, reproducibility and clarity (Required)): To elucidate whether humoral immunity and/or genetic polymorphisms contribute to the protection against P. vivax blood-stage infection, the Authors assessed whether P. vivax straintranscendent immunity can be achieved by repeated infection in Aotus monkeys.They infected six Aotus monkeys with blood stages of the P. vivax Salvador 1 (SAL-1) strain until obtaining sterile immunity, and then challenged with the heterologous AMRU-1 strain.Sterile immunity was achieved after two homologous infections, and partial protection against a heterologous AMRU-1 challenge was also achieved.IgG levels against parasite lysate by ELISA and protein microarray increased with repeated infections and correlated with the level of homologous protection.Although there were transcriptional differences in the P. vivax gene repertoire between SAL-1 and AMRU-1, there was no evidence of major antigenic switching upon homologous or heterologous challenge.These findings suggest that the partial protection observed during heterologous challenge is caused by genetic polymorphism between strains, rather than immune evasion by antigenic switching.

Full Revision
There are several non-human primate models, therefore, please specify "Aotus monkey model" in the title.

Concur:
We have added "Aotus monkey model" to the title.
2) Protein array Lines 373-374 "against major immunogenic blood stage antigens (Ags)" Please add selection criteria for how they select these 244 antigens.
We have added the following paragraph in the methods to address this comment: "All P. vivax sequences for the array used in this study were derived from the SAL-1 strain, which allowed the evaluation of greater breadth of antigens (but limited evaluation of antigenic variation).Antigens on this array were down selected from larger arrays probed with reactive sera derived from various endemic regions.Only antigens demonstrating seroreactivity across all tested sera were included [105]." They prepared protein array (n=244) based on the SAL-1 sequence.Please add a discussion of how the data was affected by the sequence difference between SAL-1 and AMRU-1 strains.They described this point only on the top 7 targets (Lines 283-287).Any further difference in antibody reactivity between polymorphic and conserved antigens (SAL-1 and AMRU-1).
We agree with this concern and have added two sentences to the discussion.
"In comparing the SAL-1 and AMRU-1 strains to the PvP01 reference strain, the sequence data demonstrated clear differences between the isolates in the whole genome analysis.Therefore, this suggests that the current iteration of the microarray (n=244) used in the study did not capture the sequence target(s) responsible for the partial protection observed." Please also add a discussion on how they can interpret their protein microarray data because the E. coli-based IVTT proteins array detects antibody responses against linear epitopes of the printed antigens.
The IVTT cell-free E.coli express system used to generate the protein microarrays represents an unbiased systems biology approach to antigen identification (Davies DH et al PMID: 26428458).The focus is intentionally on linear epitopes as attempting to capture correctly folded whole proteins is a notoriously difficult venture (Vedadi M et al Mol Biochem Parasitol. PMID: 17125854;Mehlin C et al. Mol Biochem Parasitol. PMID: 16644028).The system has shown proven utility across several disease in identifying important antigenic targets which can then be explored in greater detail using other methods (Wager LE et al. Nat Med. PMID: 33432170; Full Revision Nakajima R et al. mSphere PMID: 30541779;Virgil A et al. Future Microbiol. PMID: 20143947;Vankatesh A et al Sci Rep. PMID: 35654904).
The following text and references have been included into the discussion: "This approach was supported by previous studies which demonstrated the utility of the IVTT platform in high throughput antigen discovery across several disease areas (Jan S et al. Front Immunol PMID: 37533862;Nakajima R et al. mSphere PMID: 30541779;King CL et al. Am J Trop Med Hyg PMID: 26259938;Vankatesh A et al. Methods Mol Biol. PMID: 34115357;Vankatesh A et al. Malar J PMID: 30995911)." 3) Weakness Please summarize the weak points of this study (i.e.small number of animals used) in the Discussion section.
We have added and combined a few phrases with limitations in the discussion section: "The partial protection observed in the heterologous AMRU-1 challenges may therefore be due to major genetic differences and hence antibody epitope variation between the two strains [50].In comparing the SAL-1 and AMRU-1 strains to the PvP01 reference strain, the sequence data demonstrated clear differences between the isolates in the whole genome analysis.Therefore, this suggests that the current iteration of the microarray (n=244) used in the study did not capture the sequence target(s) responsible for the partial protection observed.To overcome this limitation and induce high levels of protective antibodies, we propose use of an immunization regime with whole parasite antigen pools from a mixture of genetically diverse strains.Another limitation of this study is the small number of subjects.The study can be considered as exploratory (i.e.looking for patterns of response rather than hypothesis testing [95]), hence the number of subjects used in the only group studied is typical of such exploratory research with humans [35,96] and NHP [38]." Minor Comments: 4) Line 129 "inoculation level II" Please reword this to "2nd inoculation" throughout the manuscript because "inoculation level" is a bit confusing for the readers.
Do not concur: It is easier to understand, in the figures in particular.Unless the editor insists, we would rather keep as is.
5) Line 320 "pir genes" Please spell out because this is the first appearance in this manuscript.

Done. Plasmodium interspersed repeat (PIR) genes.
6) Line 373 "IVTT" Please spell out because this is the first appearance in this manuscript.

Full Revision
Done. in vitro transcription/translation reaction (IVTT) 7) Line 404 "VIR antigens" Please spell out because this is the first appearance in this manuscript.
This paragraph has been removed as the data are not included in this study.
14) Line 1099 "core genes" Please add a description of what core genes mean.

Has now been added in the text line 319.
15) Figure S2 Please label each panel in Figure S2 A&B.Maybe I, II, III, IV from the left.Please also revise the label of the X-axis in Figure S2C because "Inoculation level" is misleading.
We have added the labeling to S2A and B. **Referees cross-commenting** I agree with Reviewer#2 comments.

Full Revision
1) General assessment: This is a valuable and important study conducted by qualified experts in this research field.All the works were carefully designed, and clearly presented, and the manuscript is well written.
(1) Strongest and most important aspects?Aotus monkey study with intensive data acquisition including humoral immune response and detailed parasite transcriptomic investigation.
(2) Weakness The number of animals used is rather small.
2) Advance: Does the study extend the knowledge in the field and in which way?Not only the humoral immune responses but also parasite transcriptomics data is examined for the first time.
3) Audience: Malariologists will be interested in or influenced by this research The data in this study will be the basis of future whole-parasite-based vaccine development.
My field of expertise is malariology and malaria vaccine research.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): This study focuses on the development of the model which can be further used as the model for developing a vaccine for the malaria parasite Plasmodium vivax.The researchers infected Aotus monkeys with one strain, achieved immunity, and then exposed them to a different strain.Four monkeys became immune to the initial strain, and three showed partial protection against the second strain.The researchers found that differences in genetic diversity and gene expression between strains are responsible for the varying levels of protection.This study provides insights for testing candidate vaccines against P. vivax.This model is unique and important for facilitating vaccine developments.
-The researchers provide a clear methodology and suitable for the proposed research questions.
-Did researchers observed any gametocytes after inoculations especially in the asymptomatic one or the prolong parasitemia.If they found, whether those gametocyte are infectious?
We did not focus on gametocytes in this study, hence no mosquito infection experiments were performed.

Full Revision
Reviewer #2 (Significance (Required)): The asymptomatic infections are common in malaria endemic areas but it is hard to identify the underlying immune mechanism in response to the disease.This model tries to mimic the infection in nature by reinfecting the Aotus monkeys with different stains of the parasite and then assesses the immune response with main emphasis on antibody response to the infection.This model is important to facilitate vaccine development and understanding the immune response against particular vaccines.Thank you for submitting your revised manuscript entitled "Sterile protection against Plasmodium vivax malaria by repeated blood stage infection in the Aotus monkey model".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for submitting your Research Article entitled "Sterile protection against vivax malaria by repeated blood stage infection in the Aotus monkey model".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.